Organization and Postembryonic Development of Glial Cells in the Adult Central Brain of Drosophila

13742 • The Journal of Neuroscience, December 17, 2008 • 28(51):13742–13753

Development/Plasticity/Repair Organization and Postembryonic Development of Glial Cells in the Adult Central Brain of Drosophila

Takeshi Awasaki,1* Sen-Lin Lai,1* Kei Ito,2 and Tzumin Lee1 1Department of Neurobiology, University of Massachusetts, Worcester, Massachusetts 01605, and 2Institute of Molecular and Cellular Biosciences, University of Tokyo, Tokyo 113-0032, Japan

Glial cells exist throughout the nervous system, and play essential roles in various aspects of neural development and function. Distinct types of glia may govern diverse glial functions. To determine the roles of glia requires systematic characterization of glia diversity and development. In the adult Drosophila central brain, we identify five different types of glia based on its location, morphology, marker expression, and development. Perineurial and subperineurial glia reside in two separate single-cell layers on the brain surface, cortex glia form a glial mesh in the brain cortex where neuronal cell bodies reside, while ensheathing and astrocyte-like glia enwrap and infiltrate into neuropils, respectively. Clonal analysis reveals that distinct glial types derive from different precursors, and that most adult perineurial, ensheathing, and astrocyte-like glia are produced after embryogenesis. Notably, perineurial glial cells are made locally on the brain surface without the involvement of gcm (glial cell missing). In contrast, the widespread ensheathing and astrocyte-like glia derive from specific brain regions in a gcm-dependent manner. This study documents glia diversity in the adult fly brain and demonstrates involvement of different developmental programs in the derivation of distinct types of glia. It lays an essential foundation for studying glia development and function in the Drosophila brain. Key words: glial cells; glial subtype; cell lineage; adult brain; MARCM; Drosophila

Introduction model system for studying glial development and function up to Proper development and function of neural circuitry require glial individual cells. Based on the position of glial cell bodies and cells. Although glial cells were originally believed to have just morphology, glial cells in the Drosophila ventral ganglion are supporting roles in nourishing and insulating neurons, recent classified into three types; surface-, cortex-, and neuropil- studies have revealed that glia contribute to virtually all aspects of associated glial cells (Ito et al., 1995). They are derived from nervous system development and function [for review, see Lemke specific neuroglioblasts and glioblasts (Schmidt et al., 1997; (2001), Villegas et al. (2003), and Parker and Auld (2006)]. How- Schmid et al., 1999). The glial cell missing (gcm) gene governs the ever, our understanding of glial diversity and origin is far behind derivation of all embryonic glial cells except for one specific type their neuronal counterparts. of glia, midline glia (Hosoya et al., 1995; Jones et al., 1995; Vin- Drosophila, with its accessibility to genetic, molecular, and cent et al., 1996). Loss of gcm causes deficits in glial production behavior analysis, has emerged as a powerful model system for while ectopic GCM transform neurons to glia. Thus, gcm is nec- studying brain development and function and its underlying mo- essary and sufficient for driving glial development during lecular mechanisms. In contrast to vertebrates, in which neurons embryogenesis. are outnumbered by glial cells, Drosophila have fewer neurons Embryonic glia also play essential roles in regulating neuronal and exhibits a lower glia–neuron ratio with even fewer glial cells viability and morphogenesis. Neuronal loss and connectivity de- (Ito et al., 1995; Pfrieger and Barres, 1995). fects are observed following ablation of glial cells (Booth et al., The larval ventral ganglion constitutes a relatively simple 2000; Hidalgo and Booth, 2000), although survival and func- tional differentiation of glial cells also require signals from neurons (Hidalgo et al., 2001). In addition, glial cells located at the Received Oct. 8, 2008; revised Oct. 28, 2008; accepted Nov. 1, 2008. midline, midline glia, secrete and present molecules to guide the This work was supported by Naito Foundation to T.A., a Grant-in-Aid for Scientific Research from the Ministry of growth cone of axons (Kidd et al., 1999). Education, Culture, Sports, Science, and Technology of Japan to K.I., and the United States National Institutes of Defects in glial function affect various adult behaviors of Health to T.L. We are grateful to M. R. Freeman for communicating results before publication; M. R. Freeman and Drosophila, including circadian rhythm, courtship behavior, Bloomington Stock Center for fly strains; G. G. Technau and Developmental Studies Hybridoma Bank for antibodies; H.-H. Yu for sharing UAS-mCD8::RFP strains before publication; the members of Ito’s laboratory for screening of fly and longevity (Ewer et al., 1992; Buchanan and Benzer, 1993; strains; members of Lee’s laboratory for fruitful discussion; and B. Leung for critical reading of this manuscript. Kretzschmar et al., 1997; Suh and Jackson, 2007; Grosjean et al., *T.A. and S.-L.L. contributed equally to this study. 2008). Disruption of glial function in mature brains also induces Correspondence should be addressed to either Takeshi Awasaki or Tzumin Lee at the above address. E-mail: neural degeneration (Xiong and Montell, 1995; Kretzschmar et [email protected] or [email protected]. Sen-Lin Lai’s present address: Institute of Neuroscience, University of Oregon, Eugene, Oregon 97403. al., 1997). In addition, glia play an essential role in the clearance DOI:10.1523/JNEUROSCI.4844-08.2008 of degenerating axons in both pupal and adult brains (Awasaki Copyright © 2008 Society for Neuroscience 0270-6474/08/2813742-12$15.00/0 and Ito, 2004; Watts et al., 2004; Awasaki et al., 2006; Hoopfer et Awasaki et al. • Glial Cells in Adult Drosophila Brain J. Neurosci., December 17, 2008 • 28(51):13742–13753 • 13743 al., 2006; MacDonald et al., 2006). Further elucidation of these Results glial functions and their underlying mechanisms requires better Classification of Repo-positive glial cells in the adult characterization of glial cells in the adult brain and their deriva- Drosophila central brain tion through postembryonic development of the Drosophila In the Drosophila embryonic CNS, glial cells are classified into brain. In this study, we show five different types of glial cells three categories based on the position of their cell bodies, cell locate in the adult fly brain with specific patterns and are pro- morphology, and characteristic labeling pattern of the GAL4 en- duced by distinct developmental programs. hancer trap lines: surface-, cortex-, and neuropil-associated glia (Ito et al., 1995). Except for midline glial cells, which are finally eliminated by apoptosis during the pupal stage, all other glial cells Materials and Methods in the CNS express a homeodomain protein, Repo (Xiong et al., Fly strains. To label specific subtypes of glia, we screened in total ϳ3500 1994; Halter et al., 1995; Awad and Truman, 1997). To analyze GAL4 enhancer trap strains made by NP consortium (NP lines) (Hayashi glial cells in the adult central brain, we therefore focused on the et al., 2002): NP6293 (perineurial glia), NP2276 (subperineurial glia), Repo-positive glial cells. Nuclei of Repo-positive glial cells were NP577 and NP2222 (cortex glia), NP3233 and NP1243 (astrocyte-like observed on the brain surface, in the cortex, and around and glia), and NP6520 (ensheathing glia). Note that although these lines label within neuropils (Fig. 1A,D–F). In addition, when glial mem- specific subtypes of glia preferentially, they are not pure lines exclusively brane were revealed with membrane-tagged GFP (mCD8::GFP) labeling only specific subtype of glia; every line also labels a subset of driven by repo-GAL4, surface, cortex, and neuropils of the adult neurons. NP1243 labels ensheathing glia and cortex glia weakly and brain were labeled (Fig. 1B,C). These observations suggest that NP6520 also labels cortex glia weakly. Two different repo-GAL4 lines were used as pan-glial lines localized on X and third chromosome, respectively glial cells in the adult central brain could also be classified into (Sepp et al., 2001; Lai and Lee, 2006). gcm⌬P1 (Jones et al., 1995) and three categories like those of the larval CNS. Df(2L)132 (Kammerer and Giangrande, 2001) were used for gcm mutant To examine their morphologies, we labeled small subsets of and deletion chromosome uncovering gcm and gcm2, respectively. To glial cells in the adult brain using a UAS-FLP-out system com- make a repo-GAL80 line, GAL80 from tubP-GAL80 (Lee and Luo, 1999) bined with repo-GAL4. On the brain surface, two morphologi- was subcloned into pCaSpeR4 vector, which carries the repo-promoter cally different types of glial cells were identified (Fig. 1G,H). One (Lai and Lee, 2006), and transgenic flies were generated with this con- was a small and slender oblong cell and the other was a large struct. (1) hs-FLP; UAS-FRT-rCD2-FRT-mCD8::GFP was used for FLP- sheet-like cell. These two types of glial cells were present throughout labeling. (2) hs-FLP; FRTG13, tubP-GAL80, repo-GAL80/CyO, (3) out the entire brain surface. In the cortex region, cells with mesh- repo-GAL4; G13, UAS-mCD8::GFP/CyO, and (4) repo-GAL4; G13, UAS- like morphology were evident (Fig. 1I). Each mesh-like cell oc- nlsGFP/CyO were used for systematic MARCM analysis of glia. (5) gcm- cupies a distinct part of brain cortex. Although cells labeled in the GAL4, FRTG13, UAS-nlsGFP/CyO was used for MARCM analysis of brain cortex possessed a common mesh-like structure, the size of gcm-expressing cells. (6) repo-GAL4; FRT40A, tubP-GAL80/CyO; UAS- each cell varied. In the neuropil region, two types of glial cells ⌬P1 nlsGFP/TM3 Sb, (7) hs-FLP; FRT40A, (8) hs-FLP; FRT40A, gcm /CyO, were revealed (Fig. 1J–M). One type displayed dendritic mor- and (9) hs-FLP; FRT40A, Df(2L)132/CyO were used for examining gcm phology with tiny spines (Fig. 1L), while the other exhibited fi- mutant phenotypes in glia. (10) repo-LexA::GAD; FRTG13/CyO; brous lamellar morphology (Fig. 1M). Both types of glia were UAS-mCD8::RFP, LexA-operatot-rCD2::GFP/TM3 Sb and (11) hs-FLP; found in most, if not all, neuropils. FRTG13, tubP-GAL80, repo-GAL80/CyO; NP6520/TM3 Sb were used for Based on their location and characteristic morphology, these dual-expression control MARCM analysis. Immunohistochemistry. Brains were dissected, fixed, and processed as glial cells are classified as surface, cortex, and neuropil glial cells of described previously (Lee and Luo, 1999; Ito et al., 2003). Antibodies the adult brain, respectively. Further, we identified two morpho- used in this study include rat anti-Elav (DSHB; Developmental Studies logically distinct types of surface and neuropil glia and one type of Hybridoma Bank), 1:200; mouse anti-Repo (DSHB), 1:100; rabbit anti- cortex glia in the adult central brain. Repo (Halter et al., 1995) (gift from G. M. Technau, University of Mainz, Mainz, Germany), 1:600; mouse monoclonal nc82 (anti-Bruchpilot, Organization of each type of glial cells in the adult DSHB), 1:20; rat anti-mCD8 (Caltag), 1:100; rabbit anti-GFP (Invitro- central brain gen), 1:1000; Alexa-488-, Alexa-568-, or Alexa-647-conjugated anti- In the embryonic CNS, enhancer-trap lines that label specific mouse, -rabbit, and -rat antibody (Invitrogen), 1:1000. types of glial cells have been identified (Ito et al., 1995; Becker- FLP-out and MARCM analysis. Details of FLP-out, standard MARCM, vordersandforth et al., 2008). Several genes selectively expressed and dual-expression control MARCM labeling were shown in Wong et in specific classes of embryonic glia are known (Beckervorder- al. (2002), Lee and Luo (1999), and Lai and Lee (2006), respectively. To sandforth et al., 2008). We reasoned that a given type of adult glia label cells with the FLP-out system, pupae possessing hs-FLP, FLP-out should also share a specific gene expression pattern. To target cassette, and appropriate GAL4 were incubated at 32°C for 20 min and adult glia for better characterization of class-specific organiza- labeled cells were examined in adult brain. To perform MARCM analysis, tion, development, and function, we searched for GAL4 newly hatched larvae were collected fora4hinterval and cultured at the enhancer-trap lines that label each type of glial cells in the adult density of 60–80 larvae per vial at 18°C. Mitotic recombination was fly brain. induced by heat shock at 38°C for 15 min at certain developmental peri- We identified two GAL4 lines that preferentially label the two ods and animals were raised at 18°C after heat shock. One, three, and five different types of surface glial cells (Fig. 2A,D). Although both days after larval hatching larvae at 18°C were used as mid-first-, mid- second-, and early third-instar larvae, respectively. Pupae of 1 and 3 d lines express GAL4 in the cells throughout the entire brain sur- after puparium formation at 18°C were used as early pupae and midpu- face, the distribution of the cell nuclei is quite different; one labels pae, respectively. cell nuclei densely on the brain surface (Fig. 2A) and the other Microscopic observation. Confocal serial scanning images were ob- labels cell nuclei sparsely (Fig. 2D). When single cells within each tained at 1.0 or 1.5 ␮m intervals using LSM510 laser microscope (Carl group of GAL4-expressing cells were labeled with the UAS-FLP- Zeiss). To reconstruct the stack of images, serial sections were projected out system, a small and slender oblong cell and a large sheet-like using LSM image Browser V3.1 software (Carl Zeiss). Resulting images cell were labeled, respectively (Fig. 2B,E). Their nuclei are all were processed with Photoshop CS software (Adobe Systems). positive for Repo expression (Fig. 2C,F, arrows). These results 13744 • J. Neurosci., December 17, 2008 • 28(51):13742–13753 Awasaki et al. • Glial Cells in Adult Drosophila Brain indicate that the small oblong and the large sheet-like glial cells correspond to the two types of surface glia that have been independently revealed through the above single-cell analysis of repo-GAL4-positive adult glia. Notably, these distinct types of surface glia form separate glial layers on the adult brain surface. To determine the spatial relationship of these two separate glial layers, we located GAL4-negative, Repo-positive surface nuclei in brains with only one surface glial layer selectively labeled using these subtype-specific glial GAL4 drivers (Fig. 2C,F, arrowheads). When small oblong surface glial cells were marked, Repo- positive, GFP-negative surface nuclei consistently reside underneath the layer of GFP-positive small glial cells (Fig. 2C). Al- ternatively, the arrangement of GFP- negative, Repo-positive glial nuclei situ- ated above the large GFP-labeled glial layer resembled the arrangement of small oblong glial nuclei (Fig. 2F). These results indicate that the small oblong glia form the apical glial layer and the large sheet-like glia constitute the basal glial layer on the adult brain surface. Thus, they correspond to the perineurial and subperineurial glial cells of the adult brain, respectively. We found GAL4 lines that label glia throughout the entire brain cortex (Fig. 2G). Cells with mesh-like morphology that look indistinguishable from one another were labeled following FLP-out-activation of a UAS-reporter gene in single cells with these GAL4 lines (Fig. 2I, analogous to Fig. 1I). Despite recovery of multiple independent cortex glial GAL4 enhancer-trap lines, we failed to identify lines that label cells in only subregions of the brain cortex. Thus, the glial mesh of the brain cortex is Figure 1. Repo-positive glial cells in the adult central brain. A, Glial nuclei labeled by anti-Repo antibody (green), neuronal probably composed of a single type of glial nuclei labeled by anti-Elav (blue), and neuropil labeled by nc82 (magenta). B, C, Glial membrane labeled with mCD8::GFP driven cells, which elaborate locally and together by repo-GAL4 on brain surface (B) and inner brain (C). Arrow and arrowhead show glial membrane forming borders among constitute the entire cortex mesh. When substructuresofneuropilandbraincortex,respectively.D–F,Glialnuclei(green)onbrainsurface(D),cortex(E),andneuropil(F). Glialnuclei(arrowsinDandE),neuronalnuclei,andneuropilwerelabeledasshown(A).G–I,Singlecellsofsmallsurfaceglia(G), neuronal cells were marked with anti-Elav large surface glia (H), and cortex glia (I) labeled by FLP-out combined with repo-GAL4. J–M, Single cells of neuropil glia labeled antibody, neuronal cell bodies were ar- byFLP-outcombinedwithrepo-GAL4.Adendriticandfibrouslamellarmorphologyofneuropilgliawaslabeledaroundneuropil(J).Two ranged in the holes formed by the mesh- differenttypesofneuropilgliaarrangednearby(squareinJ)areshownatahighmagnification(K).L,M,High-magnificationimagesofa like glial processes (Fig. 2H,I), suggesting dendritic (L) and fibrous lamellar morphology (M) of neuropil glia. Scale bars: A–J,50␮m; K–M,25␮m. that the cortex glia enwrap individual neuronal cell bodies in the adult brain and one pils surrounds neural bundles like the great commissure and an- cortex glial cell can enwrap many neuronal cell bodies. tennocerebral tracts (data not shown). We also identified several GAL4 enhancer-trap lines that selectively mark glial cells associating with neuropils (Fig. 3). When single cells of each type were labeled using the UAS- Among them, two patterns of labeling are discernible. Whereas FLP-out system, we obtained cells with either fibrous lamellar or cell bodies of both types are consistently located on the interface dendritic morphology (Fig. 3C,D,G,H). Nuclei of both types of between neuropil and cortex regions or among neuropil subre- cells were also labeled with anti-Repo antibody (Fig. 3C,G). These gions (Fig. 3A,E), their membranous processes elaborate differ- indicate that the fibrous lamellar and dendritic glial cells are iden- entially. One preferentially outlines the neuropils or their sub- tical to the two types of neuropil glia labeled by repo-GAL4 we compartments (Fig. 3A,B), while the other apparently fills the described earlier (compare Figs. 1L,M and 3C,G). Whereas the interior of the neuropils (Fig. 3F). Both types of glia are found in fibrous type of glia extends processes along the outer surface of all neuropil areas, including the fan-shaped body and antennal neuropil, the dendritic type elaborates inside the neuropil (Fig. lobe (Fig. 3I–L). In addition, the glial type that ensheaths neuro- 3D,H). Judging from their morphologies, the fibrous glia possi- Awasaki et al. • Glial Cells in Adult Drosophila Brain J. Neurosci., December 17, 2008 • 28(51):13742–13753 • 13745

the patterns of clones induced at different developmental times then allows one to describe the lineage relationship and further determine when a particular precur- sor is proliferating. To understand the postembryonic development of adult glia, we targeted Repo- positive adult brain cells for systematic MARCM analysis using repo-GAL4.We induced mitotic recombination with brief heat-shock pulses at various time points through postembryonic development and examined the glial cells labeled in the adult brain. Based on the morphology and position of labeled glial cells, we determined what types of glial cells were labeled. Al- though two different types of surface glia, perineurial and subperineurial glia, are readily distinguishable by size and morphology, two types of neuropil glia, ensheathing and astrocyte-like glia, are more difficult to distinguish by morphology alone when both are labeled closely and densely. Thus, in this set of experiments, we classified labeled glia into four types: perineurial, subperineurial, cortex, and neuropil glia. We determined their lineage relationship of subtypes of neuropil glia with more sophisticated MARCM techniques (see later section). Perineurial and neuropil glial cells were Figure2. Surfaceandcortexgliaofadultbrain.A–F,Perineurialglia(A–C,green)andsubperineurialglia(D–F,green)labeled frequently labeled in adult brains after withUAS-GFP(A,C,D,F)andUAS-FRT-mCD8::GFP(FLP-out)(B,E),drivenbyNP6293(A–C)andNP2276(D–F),respectively.Glial brief induction of mitotic recombination nuclei and neuronal nuclei were labeled with anti-Repo (magenta) and anti-Elav (blue), respectively. Note that GAL4-negative, during larval and pupal stages (Fig. 4A–L). Repo-positive surface nuclei (arrowheads, C and F) were labeled under and over the layer of perineurial glia and subperineurial In contrast, the subperineurial and cortex glia, respectively (arrows in C and F). G–I, Cortex glial cells (green) labeled with UAS-GFP driven by NP2222 (G, H) and with FLP-outcombinedwithNP2222(I).Neuropilswerelabeledwithnc82(magentainG).Glialnuclei(magenta)andneuronalnuclei glial cells were rarely labeled following in- (blue) were labeled with anti-Repo and anti-Elav, respectively (H, I). Arrows show nuclei of cortex glia. Scale bars, 50 ␮m. duction at various larval or pupal stages (data not shown). These results suggest that the perineurial and neuropil glial cells bly provides insulation among neighboring neural compart- are extensively generated during postembryonic development, ments and the dendritic ones potentially associate with synapses whereas the majority of subperineurial and cortex glial cells are to modulate neuronal connections similar to vertebrate astro- possibly made before larval hatching. cytes. Thus, we name them ensheathing glia and astrocyte-like Interestingly, perineurial and neuropil glial cells were labeled glia, respectively. separately with characteristic patterns. First, with mild heat shock From our screening of GAL4 enhancer-trap lines, we have at the first-instar larval stage, one could readily obtain mosaic identified several lines that preferentially label each type of glial brains that exclusively carried clones consisting of only perineur- cells in the adult brain. The distribution pattern of each GAL4 ial or neuropil glial cells (Fig. 4A,H). This suggests distinct sub- line suggests that each type of glial cells has a different function sets of larval precursors in the production of perineurial or neu- within the adult brain. Since the expression of enhancer trap lines ropil glia (Fig. 4A,H). Second, labeled glial cells were arranged reflects endogenous gene regulation, the identification of these differentially depending on the induction timing of the mitotic subtype-specific glial enhancer-trap lines suggests that each type recombination (Fig. 4A–L). Whereas large clusters of perineurial of glial cell possesses unique sets of gene expression and molecu- glial cells were labeled following induction of mitotic recombina- lar features. tion in the first-instar larvae, smaller clusters were obtained by analogous induction at later larval stages (Fig. 4, compare A,B Clonal analysis of adult glial cells to determine their and C,D). As for the neuropil glia, locally arranged clusters were developmental origins also labeled following mild heat shock before the wandering lar- Holometabolous insects undergo metamorphosis, and most of val stage (Fig. 4G–J). Labeled cells tended to form cell clusters, their adult tissues, including the brain, are generated after larval suggesting that cells of the same clonal origin do not migrate hatching. Clonal analysis by MARCM, a positive-labeling genetic randomly. Thus, many of the glial clusters are potentially derived mosaic technique, has greatly facilitated the analysis of postem- from a single progenitor. The clustering phenomenon was no bryonic neurogenesis. Briefly, using a subtype-specific GAL4 longer evident with mitotic recombination induced at the wan- driver, one can target a specific subset of brain cells for labeling by dering larval or early pupal stages. In these mosaic brains, al- inducing mitotic recombination in their precursors. Analysis of though many labeled perineurial and neuropil glia remained, 13746 • J. Neurosci., December 17, 2008 • 28(51):13742–13753 Awasaki et al. • Glial Cells in Adult Drosophila Brain

Figure 3. Two subtypes of neuropil glia of adult central brain. Ensheathing (A–D, I, K) and astrocyte-like (E–H, J, L) glia labeled with NP1243 and NP6520, respectively. Entire and single cells of neuropil glial subtype were labeled with UAS-GFP (A, E, and I–L), UAS-mCD8::GFP (B, F), and UAS-FLP-out (C, D, G, H), respectively. Arrows show nuclei of each subtype of glial cells labeled with anti-Repo (magenta in C, G, and I–L). Neuronal nuclei (blue) were labeled with anti-Elav (B, F, K, L) and neuropil (magenta) was labeled with nc82 (D, H). Scale bars, 50 ␮m. they were widely distributed and mostly present singly or in very while marking the whole cells. We identified all the clusters and small clusters (Fig. 4E,F,K,L). Finally, labeled cells were rarely determined the size of each cluster in the adult brains undergoing obtained when mitotic recombination was induced after midpu- somatic recombination at specific developmental time points pal development (data not shown). Given the independent ori- (Fig. 4M,N,R). We defined a single cluster as a group of cells gins for the different types of adult glia, quantitative analyses of among which physical contact can extend from one to another, the stage-specific clone inductions were conducted separately for and counted the number of nuclei within one cluster as its cluster the perineurial glia and the neuropil glia. size. If cells proliferate exponentially, the cluster size should decrease exponentially as clones are induced at later time points Postembryonic development of perineurial glial cells while the total cluster number should increase drastically as the Postmitotic cells might increase exponentially through multiple pool of dividing glia expanded with time. On the other hand, if rounds of symmetric divisions or derive sequentially from self- cells proliferate by asymmetric division of self-renewing progen- renewing progenitors via asymmetric divisions. The analysis of itors, the cluster size should decrease gradually as clones are in- cluster size distribution should help distinguish between these duced at later time points while the total cluster number should two possibilities. For example, one would expect production of not change because the number of self-renewing progenitors re- single-cell or two-cell clones through the entire process from mains constant. Consistent with the predictions made based on asymmetric divisions or only near the end of gliogenesis from an exponential model of cell proliferation, we obtained a few symmetric division depending on when the terminal mitoses for large clusters of glia following clone induction in early larval mature glial cells occurred. However, we found it challenging to stages versus many small clusters of glia in the brains that received count glial cell numbers when they were labeled with a membrane heat shock at late larval stages (Fig. 4M,N). Furthermore, we marker, because, unlike neurons, glial cells lack cell-body do- rarely obtained single-cell clones of glia except when mitotic remains and closely contact with one another. combination was induced in wandering larvae or early pupae To better quantify the sizes of glial clusters, we repeated the before the end of gliogenesis around the midpupal stage when the stage-specific induction of MARCM clones and resorted to frequency of clones reduced to a background level (Fig. 4R). nuclear-GFP, driven by repo-GAL4, for highlighting glial nuclei These results suggest that perineurial glial cells are made through Awasaki et al. • Glial Cells in Adult Drosophila Brain J. Neurosci., December 17, 2008 • 28(51):13742–13753 • 13747

Figure 4. Systematic MARCM analysis of glial proliferation during postembryonic development. A–L, Perineurial (A–F) and neuropil (G–L) glial cells labeled by heat-shock-induced mitotic recombination in mid-first-instar larvae (A, B), newly hatched larvae (G, H), early third-instar larvae (C, D, I, J) and early pupae (E, F, K, L). Scale bar, 50 ␮m. M–R, Quantification of glial cells that were labeled by MARCM system with heat-shock-induced mitotic recombination at different developmental stages (x-axis). Average number (M, O) and size (N, P) of perineurial glia (M, N) and neuropilglia(O,P)clustersthatwereinducedatdifferentdevelopmentalstage( y-axis).Q,Averagenumberoflargeperineurialglia(Ͼ10cells)andneuropilglia(Ͼ8)clustersperbrain.R,Average number of single cell clones of perineurial and neuropil glia. Same samples were used for analysis in M–R. Numbers of examined brains are shown in M. Genotypes: hs-FLP/repo-GAL4; FRTG13, UAS-mCD8::GFP/FRTG13, tubP-GAL80, repo-GAL80 (A–L) and hs-FLP/repo-GAL4; FRTG13, UAS-nlsGFP/FRTG13, tubP-GAL80, repo-GAL80 (M–R). exponential cell proliferation, and that most perineurial glial pre- gliogenesis continues until the midpupal stage, perineurial glia cursors enter the final round of proliferation around the wander- probably only exist as precursors in the developing Drosophila ing larval or early pupal stage. nervous system. To locate such perineurial glial precursors, we Given that most, if not all, of the adult perineurial glial cells are examined the lineages of proliferating perineurial glia in wander- born after larval hatching and that this postembryonic process of ing larvae. Since mitotic recombination at the mid-first-instar 13748 • J. Neurosci., December 17, 2008 • 28(51):13742–13753 Awasaki et al. • Glial Cells in Adult Drosophila Brain larval stage could label large perineurial glial clusters in the adult brain (Fig. 4N), we induced mitotic recombination at this stage and examined the labeled cells in the wandering larvae. Cell clusters covering the surface of the larval brain were observed by this labeling (Fig. 5A,B). Whereas the perineurial glia have slender and oblong morphology extending along the dorsoventral axis in the adult brain (Figs. 1G,2B,5C), these larval cells extend filopodial processes radially (Fig. 5B, arrows), indicating that they are the mor- phologically immature perineurial glia of the adult. These results suggest that perineurial glia proliferate and mature on the brain surface locally and expand as the brain surface enlarges during development.

Postembryonic development of neuropil glial cells We also analyzed postembryonic proliferation of neuropil glia with MARCM (Fig. 4O–R). Although more neuropil glial clusters were obtained with mitotic recombination induced at later developmental stages (Fig. 4O), there was no significant change in the average size of clusters generated at different times (Fig. 4P). Large clusters (Ͼ8 cells) could be induced at comparable frequencies through larval development except mid-first larval stage (Fig. 4Q). In contrast, the frequency in ob- taining clusters with Ͼ10 perineurial glial cells reduced drastically after early larval stages (Fig. 4Q). In addition, the average cell number per cluster of neuropil glia was smaller than that of the perineurial glial cells (Fig. 4, compare N, P). These results suggest that the proliferation pattern of neuropil glia is different from that of perineurial glia. However, as in perineurial Figure 5. Precursors of perineurial and neuropil glia. Perineurial glia cluster in a wandering larva (A, B) and adult (C), which werelabeledbyMARCMsystemwithheat-shock-inducedmitoticrecombinationinmid-first-instarlarvae.Arrowsshowfilopodial glia, we rarely obtained single-cell clones processes extending from larval perineurial glia. Multiple neuropil glia clusters in adults (D–F) and wandering larvae (G–L), of neuropil glia until late larval or early whichwerelabeledbyMARCMwithlongheat-shock(40min)inducedmitoticrecombinationinNHL.Notethatlabeledcellswere pupal stages (Fig. 4R). This result argues localized in posterior-medial (G, J), lateral (H, K), and dorsal (I, L) interface between brain cortex and neuropil. J–L, High- against a model where neuropil glia are magnification images of G–I, respectively. Glial nuclei were labeled with anti-Repo (magenta, A and G–I). Scale bars, 50 ␮m. generated by asymmetric cell division. In- stead, neuropil glia are also derived processes (Fig. 5J–L). Mature neuropil glia should reside not only through multiple rounds of symmetric cell division, but each in the neuropil–cortex interface but also among the neuropils progenitor undergoes fewer divisions than that of perineurial that lie apart from the brain cortex (Figs. 1F,3B,F). Further- glia, and the timing of proliferation appears irregular, although more, the analogously induced neuropil glial clones are more the final mitoses to derive postmitotic neuropil glia remain rather broadly distributed when examined at the adult stage (Fig. 5, synchronized around puparium formation. compare D–F and G–I). These results collectively suggest that We further examined precursors of neuropil glia in the wan- precursors of neuropil glia proliferate in the specific regions of dering larvae. Following a 40 min heat shock in NHL (newly the larval brain and migrate to maturate upon metamorphosis. hatched larvae), we consistently obtained clusters of neuropil glia in every mosaic brain (Fig. 5D–F). When such mosaic brains The function of gcm in postembryonic gliogenesis were examined at the wandering larval stage, the labeled Repo- During embryogenesis, glial cell missing (gcm) plays an essential positive cells on the interface between neuropil and cortex were role in glial development. Is gcm also essential for postembryonic consistently clustered in at least three specific regions, the pos- gliogenesis of perineurial and neuropil glia? To address this ques- teromedial, lateral, and dorsal interface (Fig. 5G–L). In addition, tion, we first examined gcm expression in the developing brain whereas mature neuropil glia acquire a fibrous lamellar or den- with gcm-GAL4. Three clusters of gcm-positive cells were found dritic morphology, these cells are round-shaped with filopodial in the interface between neuropil and cortex of the larval brain. Awasaki et al. • Glial Cells in Adult Drosophila Brain J. Neurosci., December 17, 2008 • 28(51):13742–13753 • 13749

Figure 6. Function of gcm on postembryonic development of perineurial and neuropil glia. A, B, Cells labeled with GFP driven by gcm-GAL4 in a wandering larva. C, D, Cells labeled by MARCM system with gcm-GAL4. Mitotic recombination was induced in NHL. Glial nuclei were labeled with anti-Repo antibody (magenta, A–D). High-magnification images of A and C are shown in B and D, respectively.Notethatgcm-positivecellswerelabeledwithanti-Repoantibody(B,D).E–H,Wild-type(E,G)andgcm⌬P1 (F,H)MARCMclonesofneuropilglia(E,F)inducedatNHLandperineurial glia (G, H) induced at mid-first-instar larvae, respectively. I, J, Quantification of neuropil glia (I) and perineurial glia (J) that were labeled by MARCM system. Average number of neuropil glia per brain (I) and average number of large perineurial glia cluster (Ͼ10) per brain were examined in wt, gcm⌬P1, and Df(2L)132 clones. Scale bars, 50 ␮m. Genotypes: gcm-GAL4/UAS-GFP (A, B); hs-FLP/ϩ; gcm-GAL4, FRTG13, UAS-nlsGFP/FRTG13, tubP-GAL80, repo-GAL80 (C, D); hs-FLP, UAS-mCD8::GFP/repo-GAL4; FRT40A, tubP-GAL80/FRT40A or FRT40A, gcm⌬P1 (E–H); and hs-FLP/repo- GAL4; FRT40A, tubP-GAL80/FRT40A or FRT40A, gcm⌬P1 or FRT40A, Df(2L)132; UAS-nlsGFP/ϩ (I, J).

Their morphology and location are similar to the clusters of neu- fully dispensable in perineurial glia. First, gcm⌬P1 mutant neuro- ropil glia precursors (compare Figs. 5G–L and 6A,B). gcm expres- pil glia were barely detectable even after a 40 min heat shock that sion could be detected beginning in early third instar, and it reproducibly yielded ϳ50 homozygous wild-type neuropil glial becomes prominent in the wandering larval stage (data not cells per control mosaic brain (Fig. 6E,F,I). In contrast, there was shown). The cells expressing gcm spread along the cortex–neuro- no significant difference in the cluster number or size of peri- pil interface during early pupal stage and the expression disap- neurial glia between control and gcm⌬P1 clones (Fig. 6G,H,J). pears after the midpupal stage (data not shown). In addition, cells Second, since gcm and gcm2 possess partially redundant function labeled with gcm-GAL4 in the interface are also recognized with in the glial and neuronal development of the optic lobe (Chotard anti-Repo antibody (Fig. 6B,D). Furthermore, analogous pat- et al., 2005), it is possible that gcm2 might compensate for the loss terns of neuropil glial clones were obtained in the mosaic larval of gcm in the support of perineurial glial development. To exam- brain when MARCM experiments were performed with either ine this possibility, we determined how deletion of both gcm and repo-GAL4 or gcm-GAL4 (Fig. 6C,D, compared with Fig. 5G,J). gcm2 in the Df(2L)132 deficiency (Kammerer and Giangrande, These results suggest that precursors of neuropil glia express gcm 2001) might affect the clonal development of specific adult glia. transiently. In contrast, we could not detect any cells labeled with Again, normal-looking mutant perineurial glial clones could be gcm-GAL4 on the brain surface during larval and pupal stage. In readily obtained while mutant neuropil glia were largely absent addition to the transient expression in the neuropil glial precur- (Fig. 6I,J). The effects of gcm mutations on the adult glia, to- sors, gcm-GAL4 persistently labels two clusters of neurons bilat- gether with expression pattern of gcm, suggest that gcm is essential erally in the central brain (data not shown). for the postembryonic development of the neuropil glia but not Next we determined the effect of gcm mutations on postem- for the perineurial glia. bryonic development of adult glial cells using the MARCM system with repo-GAL4. Somatic recombination was induced with The cell lineage of two different neuropil glia 40 min heat shock in NHL and with a 20 min heat shock in Perineurial and neuropil glia are apparently derived from differ- mid-first-instar larvae to generate clones of neuropil and peri- ent precursors, as evidenced by multiple independent lines of neurial glia, respectively. We obtained results that are consistent evidence, including their differential dependence on gcm. How, with the notion, as implied from the expression pattern, that gcm then, do the two subtypes of neuropil glia differentiate? We found is required for proper development of neuropil glia but appears it challenging to determine subtype identity of neuropil glia 13750 • J. Neurosci., December 17, 2008 • 28(51):13742–13753 Awasaki et al. • Glial Cells in Adult Drosophila Brain

Figure 7. Neuropil glial clones. Ensheathing glial clones (A, C, E, F) and astrocyte-like glial clones (B, D, G) labeled by standard MARCM (A–D) and dual-expression control MARCM (E–H). Note thatCandDshowglialcloneslocatedintheantennallobe.WhereastheensheathingglialclonewaslabeledbybothrCD2::GFPdrivenbyrepo-LexA::GAD(green,E)andmCD8::RFPdrivenbyNP6520 (magenta,F),theastrocyte-likeglialconewaslabeledbyonlyrCD2::GFPdrivenbyrepo-LexA::GAD(green,G),butnotbymCD8::RFPdrivenbyNP6520(magenta,H).Scalebars,50␮m.Genotypes: hs-FLP/repo-GAL4; FRTG13, UAS-mCD8::GFP/FRTG13, tubP-GAL80, repo-GAL80 (A–D) and hs-FLP/repo-LexA::GAD; FRTG13/FRTG13, tubP-GAL80, repo-GAL80; NP6520/UAS-mCD8::RFP, LexA-operator-rCD2::RFP (E–H). among cells that constitute a large cluster of neuropil glia. Al- types of neuropil glia differ not only in morphology and marker though some large clusters of neuropil glia appear homogeneous expression but also with different origins of development. consisting of either ensheathing glia or astrocyte-like glia (Fig. 7A–D), it remained unclear whether the ensheathing and Discussion astrocyte-like glia consistently arise from separate lineages. Our analysis of Repo-expressing cells reveals the presence of three To address this question, we repeated clonal analysis with classes and five types of glia in the adult Drosophila central brain. dual-expression-control MARCM that permits differential label- Each glial type exhibits a characteristic location and morphology. ing of distinct cells in a given MARCM clone owing to indepen- Multiple glial functions can be inferred from their different cel- dent controls over distinct reporter genes by GAL4 versus lular organizations in the brain. The different functions of spe- LexA::GAD, two GAL80-suppressible binary transcriptional ac- cific glial types are further supported by their differential marker tivators (Lai and Lee, 2006). We used repo-LexA::GAD plus the expression. In addition, different glial types derive independently ensheathing glial GAL4 driver, NP6520, in dual-expression- from distinct populations of precursors. They proliferate in dif- control MARCM, to label GAL80-minus ensheathing glia with ferent temporal and spatial patterns, and the involvement of gcm both lexAop-rCD2::GFP and UAS-mCD8::RFP while marking the varies from one to another glial type. Identification of glial diversity astrocyte-like glia of the same genotype with lexAop-rCD2::GFP lays an essential foundation for unraveling the diverse glial functions only. If a neuropil glial cluster consisted of only ensheathing or in the brain, and determining how diverse glial types are derived is astrocyte-like glia, one would expect expression of mCD8::RFP in integral to the understanding of how a mature brain is made. all or none of the cells in the cluster. In contrast, if ensheathing Two layers of distinct glial cells surround the entire adult Dro- and astrocyte-like glia derived from common progenitors, one sophila brain. The inner one, possibly composed of a fixed num- would detect mCD8::RFP in a subset of rCD2::GFP-marked cells ber of large sheet-like subperineurial glial cells since larval hatch- in a single cluster. Fifteen clusters of neuropil glia were obtained ing, constitutes the fly blood–brain barrier in both larval and following induction of somatic recombination at the newly adult brains (Bainton et al., 2005; Schwabe et al., 2005; Stork et hatched larval stage. Among them, 12 clusters were found to al., 2008). In contrast, the outer layer consists of numerous small purely consist of either ensheathing glia or astrocyte-like glia, as oblong perineurial glial cells that proliferate through postembry- evidenced by the expression of mCD8::RFP in all or none of the onic development of the brain and may remain immature until cells in a given cluster (Fig. 7E–H). In the other three clusters that formation of the adult brain. Given its more external location and contained both mCD8::RFP-positive and -negative cells, no mat- apparent adherence to the subperineurial layer, the perineurial glia ter whether ensheathing or astrocyte-like glia predominated, the possibly helps make up the adult blood–brain barrier. However, minority was overwhelmed and composed of only one or two these two types of surface glia likely play nonoverlapping, comple- cells per cluster despite the cluster size being roughly Ͼ8 cells. mentary roles in the regulation of the permeability to the CNS, as Since a low level of mitotic recombination could occur even with- implied from their different enhancer trap expression patterns. out any heat-shock induction (Fig. 4R), it is possible that the The other three types of glia form independent networks in- presence of one or two heterotypic cells in the otherwise homo- side the brain. Cortex glia enwrap individual neuronal cell bodies geneous population may occur as a consequence of multiple in the brain cortex. Each cortex glial cell send mesh-like processes events of mitotic recombination. Given that ensheathing and to surround multiple neuronal cell bodies locally. Cortex glia may astrocyte-like glia rarely coexist in a single cluster, the two sub- provide metabolic support for neurons and have the potential for Awasaki et al. • Glial Cells in Adult Drosophila Brain J. Neurosci., December 17, 2008 • 28(51):13742–13753 • 13751

(Sohal et al., 1972; Strausfeld, 1976), interface and neuropil glia in Acheta (Meyer et al., 1987) and neuropil cover type and neuropil glia or simple and complex neuropil glia in Manduca (Cantera, 1993; Oland et al., 1999). One type is localized cortex/ neuropil interface or borders among neuropils and ensheaths neuropil, and another type extends glial processes inside the neuropils, like the ensheathing and astrocyte-like glia of adult Drosophila brain, respectively. It is, therefore, these two subtypes of neuropil glia would com- monly exist in insects. These distinct types of adult fly brain glia arise independently from each other. The proliferation of subperineurial glia is probably restricted to embryogenesis, and any postembryonic increase in the cortex glial cell number should be much less significant than the expansion of the other three types of adult glia, which primarily occurs during larval development. Fur- thermore, distinct populations of precursors give rise to perineurial, ensheathing, or astrocyte-like glia (Fig. 8). Notably, perineurial glia precursors distribute around the brain surface and make clones that ex- Figure8. Schematicmodelofpostembryonicglialproliferation.A,B,Perineurialglia(A)andtwosubtypesofneuropilglia(B). pand locally as the brain grows in size through development. In contrast, en- modulating neuronal cell body functions in a spatially controlled sheathing and astrocyte-like glial cells are derived from specific manner. Further inside the brain lie two types of neuropil glia. proliferation centers and do not migrate throughout the brain The ensheathing glia outlines individual neuropils and their sub- until metamorphosis. Most somatic clones of neuropil glia exclu- compartments, while the astrocyte-like glia extend processes into sively consist of ensheathing or astrocyte-like glial cells. However, the neuropils where synapses form. the identity of the neuropil clones could not be unambiguously The glial boundary established by the ensheathing glia may determined until late pupal stages, making it unclear whether the provide the necessary insulation to prevent lateral communica- progenitors of the ensheathing and astrocyte-like glia are mixed tion between adjacent neuropils, and provide septa when a neu- in those proliferation centers. Perineurial glial precursors make ropil is composed of multiple independent subcompartments. In clones of similar sizes at a given developmental stage, suggesting contrast, astrocyte-like glial cells potentially associate with syn- that they retain the same proliferation potential through devel- apses. Several electron microscopic studies have shown that glial opment. In contrast, the clones of neuropil glia derived at the processes are located around synapses of insect neuropils (Carl- same stage vary in size, raising the possibility that neuropil glial son and Saint Marie, 1990). In an analogous manner, mamma- precursors undergo stochastic proliferation, possibly, in response lian astrocytes target processes to synapses. They express excita- to some limited local cue(s). Supply of Vein, a neuregulin-like tory amino-acid transporters (EAAT-1 and -2), and can recover trophic factor, from neurons has been shown to promote local excess extracellular glutamate or aspartate to keep the local envi- proliferation of the embryonic longitudinal glia (Griffiths and ronment suitable for the next synaptic transmission (Kanai, 1997; Hidalgo, 2004). It remains to be determined whether similar Takahashi et al., 1997). In addition, mammalian astrocytes may mechanisms govern the postembryonic expansion of the adult release gliotransmitters in response to synaptic activity to modu- neuropil glia. Finally, all the postembryonic glial precursors ap- late synaptic functions (Halassa et al., 2007). Whereas in the ver- parently divide symmetrically to produce two daughters follow- tebrate nervous system synapses are formed not only on the den- ing each division; intriguingly, the final round of mitosis consis- drites and axon terminals but also on the surface of the neural cell tently takes place around pupal formation. This suggests a stage- bodies, in the invertebrate brain no synapses are formed on the specific signal may trigger the final glia-producing mitoses. neuronal cell bodies (Ito and Awasaki, 2008). Accordingly, the The involvement of different developmental programs in the cortex glia and neuropil glia are associated exclusively to the neu- derivation of different types of adult glia is further evidenced in ral cell bodies and synaptic neuropils, respectively. Although the differential temporal requirement for gcm. GCM promotes all Drosophila glia also expresses EAAT-1 (dEAAT-1) that is concen- embryonic gliogenesis except for midline glia (Hosoya et al., trated in neuropils (Rival et al., 2004), it remains to be deter- 1995; Jones et al., 1995; Vincent et al., 1996). GCM suppresses mined which function of the vertebrate astrocytes is achieved by neural differentiation through expression of Tramtrack and con- which type of insect glial cells. fers glial identity via induction of Repo and other glial genes In the adult brain or ventral nerve cord of other insects, two (Jones, 2001). Transient expression of gcm also occurs in the types of glial cells associated with neuropil have been identified; postembryonic development of both medulla and thoracic glial type II and type III glia or interface and neuropilar glia in Musca lineages, and inhibition of GCM function by expression of a 13752 • J. Neurosci., December 17, 2008 • 28(51):13742–13753 Awasaki et al. • Glial Cells in Adult Drosophila Brain dominant-negative form of GCM suppresses differentiation of Ewer J, Frisch B, Hamblen-Coyle MJ, Rosbash M, Hall JC (1992) Expression their glial development (Colonques et al., 2007; Soustelle and of the period clock gene within different cell types in the brain of Drosoph- Giangrande, 2007). In addition, we show that precursors of neu- ila adults and mosaic analysis of these cells’ influence on circadian behav- ioral rhythms. J Neurosci 12:3321–3349. ropil glia express gcm transiently and the postembryonic induc- Griffiths RL, Hidalgo A (2004) Prospero maintains the mitotic potential of tion of the gcm mutant clone effectively arrest the development of glial precursors enabling them to respond to neurons. EMBO J neuropil glia. These observations indicate that GCM plays anal- 23:2440–2450. ogous roles in promoting glial fate at different developmental Grosjean Y, Grillet M, Augustin H, Ferveur JF, Featherstone DE (2008) A stages. It is possible that the precursors of neuropil glia do not glial amino-acid transporter controls synapse strength and courtship in acquire full glial identity until gcm is expressed at an intermediate Drosophila. Nat Neurosci 11:54–61. stage of development. In contrast, gcm is dispensable for postem- Halassa MM, Fellin T, Haydon PG (2007) The tripartite synapse: roles for gliotransmission in health and disease. Trends Mol Med 13:54–63. bryonic development of perineurial glia. Unlike midline glia in Halter DA, Urban J, Rickert C, Ner SS, Ito K, Travers AA, Technau GM which Repo is never expressed, perineurial glial cells are strongly (1995) The homeobox gene repo is required for the differentiation and positive for Repo, a direct target for GCM, making it likely that maintenance of glia function in the embryonic nervous system of Dro- GCM is involved in the development of perineurial glia. It ap- sophila melanogaster. Development 121:317–332. pears more plausible that like neuropil glia transient expression Hayashi S, Ito K, Sado Y, Taniguchi M, Akimoto A, Takeuchi H, Aigaki T, of gcm also occurs in the perineurial glial precursors but at an Matsuzaki F, Nakagoshi H, Tanimura T, Ueda R, Uemura T, Yoshihara earlier stage, and that they have already acquired glial cell fate M, Goto S (2002) GETDB, a database compiling expression patterns and molecular locations of a collection of Gal4 enhancer traps. Genesis before larval hatching. 34:58–61. While most glial types appear to be conserved in flies, the Hidalgo A, Booth GE (2000) Glia dictate pioneer axon trajectories in the microglia type of glia is not obvious in the adult fly brain. It is Drosophila embryonic CNS. Development 127:393–402. possible that we failed to identify all major types of glia because of Hidalgo A, Kinrade EF, Georgiou M (2001) The Drosophila neuregulin vein the restriction of our analysis to only Repo-positive cells. Never- maintains glial survival during axon guidance in the CNS. Dev Cell theless, evidence is accumulating that neuropil glia may share 1:679–690. microglia-type functions to engulf degenerating axons in the pro- Hoopfer ED, McLaughlin T, Watts RJ, Schuldiner O, O’Leary DD, Luo L (2006) Wlds protection distinguishes axon degeneration following injury from nat- cess of programmed axon pruning or following injury of neurites urally occurring developmental pruning. Neuron 50:883–895. (Awasaki and Ito, 2004) (M. R. Freeman, personal communica- Hosoya T, Takizawa K, Nitta K, Hotta Y (1995) glial cells missing: a binary tion). Additional glial diversity has been suggested in vertebrates. switch between neuronal and glial determination in Drosophila. Cell Many subtypes of glial cells in the Drosophila embryonic ventral 82:1025–1036. ganglion has also been revealed through detailed analysis of lin- Ito K, Awasaki T (2008) Clonal unit architecture of the adult fly brain. In: eage as well as gene expression (Schmidt et al., 1997; Schmid et al., Brain development in Drosophila melanogaster (Technau GM, ed), pp 1999; Beckervordersandforth et al., 2008). The identification of 137–158. Austin: Landes Bioscience. Ito K, Urban J, Technau G (1995) Distribution, classification, and develop- five major types of adult fly brain glia and the analysis of their ment of Drosophila glial cells in the late embryonic and early larval ventral developmental origins pave a way for further use of the Drosoph- nerve cord. Dev Genes Evol 204:284–307. ila as a model system for study of glial diversity, development, Ito K, Okada R, Tanaka NK, Awasaki T (2003) Cautionary observations on and function. preparing and interpreting brain images using molecular biology-based staining techniques. Microsc Res Tech 62:170–186. References Jones BW (2001) Glial cell development in the Drosophila embryo. Bioes- Awad TA, Truman JW (1997) Postembryonic development of the midline says 23:877–887. glia in the CNS of Drosophila: proliferation, programmed cell death, and Jones BW, Fetter RD, Tear G, Goodman CS (1995) glial cells missing: a endocrine regulation. Dev Biol 187:283–297. genetic switch that controls glial versus neuronal fate. Cell 82:1013–1023. Awasaki T, Ito K (2004) Engulfing action of glial cells is required for pro- Kammerer M, Giangrande A (2001) Glide2, a second glial promoting factor grammed axon pruning during Drosophila metamorphosis. Curr Biol in Drosophila melanogaster. EMBO J 20:4664–4673. 14:668–677. Kanai Y (1997) Family of neutral and acidic amino acid transporters: mo- Awasaki T, Tatsumi R, Takahashi K, Arai K, Nakanishi Y, Ueda R, Ito K lecular biology, physiology and medical implications. Curr Opin Cell Biol (2006) Essential role of the apoptotic cell engulfment genes draper and 9:565–572. ced-6 in programmed axon pruning during Drosophila metamorphosis. Kidd T, Bland KS, Goodman CS (1999) Slit is the midline repellent for the Neuron 50:855–867. robo receptor in Drosophila. Cell 96:785–794. Bainton RJ, Tsai LT, Schwabe T, DeSalvo M, Gaul U, Heberlein U (2005) Kretzschmar D, Hasan G, Sharma S, Heisenberg M, Benzer S (1997) The moody encodes two GPCRs that regulate cocaine behaviors and blood- Swiss cheese mutant causes glial hyperwrapping and brain degeneration brain barrier permeability in Drosophila. Cell 123:145–156. in Drosophila. J Neurosci 17:7425–7432. Beckervordersandforth RM, Rickert C, Altenhein B, Technau GM (2008) Lai SL, Lee T (2006) Genetic mosaic with dual binary transcriptional sys- Subtypes of glial cells in the Drosophila embryonic ventral nerve cord as tems in Drosophila. Nat Neurosci 9:703–709. related to lineage and gene expression. Mech Dev 125:542–557. Lee T, Luo L (1999) Mosaic analysis with a repressible cell marker for studies Booth GE, Kinrade EF, Hidalgo A (2000) Glia maintain follower neuron sur- of gene function in neuronal morphogenesis. Neuron 22:451–461. vival during Drosophila CNS development. Development 127:237–244. Lemke G (2001) Glial control of neuronal development. Annu Rev Neuro- Buchanan RL, Benzer S (1993) Defective glia in the Drosophila brain degen- sci 24:87–105. eration mutant drop-dead. Neuron 10:839–850. MacDonald JM, Beach MG, Porpiglia E, Sheehan AE, Watts RJ, Freeman MR Cantera R (1993) Glial cells in adult and developing prothoracic ganglion of (2006) The Drosophila cell corpse engulfment receptor Draper mediates the hawk moth Manduca sexta. Cell Tissue Res 272:93–108. glial clearance of severed axons. Neuron 50:869–881. Carlson SD, Saint Marie RL (1990) Structure and function of insect glia. Meyer MR, Reddy GR, Edwards JS (1987) Immunological probes reveal spatial Annu Rev Entomol 35:597–621. and developmental diversity in insect neuroglia. J Neurosci 7:512–521. Chotard C, Leung W, Salecker I (2005) glial cells missing and gcm2 cell Oland LA, Marrero HG, Burger I (1999) Glial cells in the developing and adult autonomously regulate both glial and neuronal development in the visual olfactory lobe of the moth Manduca sexta. Cell Tissue Res 297:527–545. system of Drosophila. Neuron 48:237–251. Parker RJ, Auld VJ (2006) Roles of glia in the Drosophila nervous system. Colonques J, Ceron J, Tejedor FJ (2007) Segregation of postembryonic neu- Semin Cell Dev Biol 17:66–77. ronal and glial lineages inferred from a mosaic analysis of the Drosophila Pfrieger FW, Barres BA (1995) What the fly’s glia tell the fly’s brain. Cell larval brain. Mech Dev 124:327–340. 83:671–674. Awasaki et al. • Glial Cells in Adult Drosophila Brain J. Neurosci., December 17, 2008 • 28(51):13742–13753 • 13753

Rival T, Soustelle L, Strambi C, Besson MT, Iche´ M, Birman S (2004) De- Organization and function of the blood–brain barrier in Drosophila. creasing glutamate buffering capacity triggers oxidative stress and neuro- J Neurosci 28:587–597. pil degeneration in the Drosophila brain. Curr Biol 14:599–605. Strausfeld NJ (1976) Insect brain. New York: Springer. Schmid A, Chiba A, Doe CQ (1999) Clonal analysis of Drosophila embry- Suh J, Jackson FR (2007) Drosophila ebony activity is required in glia for the onic neuroblasts: neural cell types, axon projections and muscle targets. circadian regulation of locomotor activity. Neuron 55:435–447. Development 126:4653–4689. Takahashi M, Billups B, Rossi D, Sarantis M, Hamann M, Attwell D (1997) Schmidt H, Rickert C, Bossing T, Vef O, Urban J, Technau GM (1997) The The role of glutamate transporters in glutamate homeostasis in the brain. embryonic central nervous system lineages of Drosophila melanogaster. II. J Exp Biol 200:401–409. Neuroblast lineages derived from the dorsal part of the neuroectoderm. Villegas SN, Poletta FA, Carri NG (2003) GLIA: A reassessment based on novel data on the developing and mature central nervous system. Cell Biol Dev Biol 189:186–204. Int 27:599–609. Schwabe T, Bainton RJ, Fetter RD, Heberlein U, Gaul U (2005) GPCR sig- Vincent S, Vonesch JL, Giangrande A (1996) Glide directs glial fate commit- naling is required for blood-brain barrier formation in Drosophila. Cell ment and cell fate switch between neurones and glia. Development 123:133–144. 122:131–139. Sepp KJ, Schulte J, Auld VJ (2001) Peripheral glia direct axon guidance Watts RJ, Schuldiner O, Perrino J, Larsen C, Luo L (2004) Glia engulf degener- across the CNS/PNS transition zone. Dev Biol 238:47–63. ating axons during developmental axon pruning. Curr Biol 14:678–684. Sohal RS, Sharma SP, Couch EF (1972) Fine structure of the neural sheath, Wong AM, Wang JW, Axel R (2002) Spatial representation of the glomeru- glia and neurons in the brain of the housefly, Musca domestica. Z Zellfor- lar map in the Drosophila protocerebrum. Cell 109:229–241. sch Mikrosk Anat 135:449–459. Xiong WC, Montell C (1995) Defective glia induce neuronal apoptosis in Soustelle L, Giangrande A (2007) Novel gcm-dependent lineages in the the repo visual system of Drosophila. Neuron 14:581–590. postembryonic nervous system of Drosophila melanogaster. Dev Dyn Xiong WC, Okano H, Patel NH, Blendy JA, Montell C (1994) repo encodes 236:2101–2108. a glial-specific homeo domain protein required in the Drosophila nervous Stork T, Engelen D, Krudewig A, Silies M, Bainton RJ, Kla¨mbt C (2008) system. Genes Dev 8:981–994.